Whales originated from aquatic artiodactyls in the Eocene Epoch of India

Abstract

Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link to the ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth. We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids were aquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.

Full text

ARTICLES
Whales originated from aquatic
artiodactyls in the Eocene epoch of India
J. G. M. Thewissen
1
, Lisa Noelle Cooper
1,2
, Mark T. Clementz
3
, Sunil Bajpai
4
& B. N. Tiwari
5
Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link to
the ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but
until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid
artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the
structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth.
We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids were
aquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.
Phylogenetic analyses of molecular data on extant animals strongly
support the notion that hippopotamids are the closest relatives of
cetaceans (whales, dolphins and porpoises)
1–3
. In spite of this, it is
unlikely that the two groups are closely related when extant and
extinct artiodactyls are analysed, for the simple reason that cetaceans
originated about 50 million years (Myr) ago in south Asia, whereas
the family Hippopotamidae is only 15 Myr old, and the first hippo-
potamids to be recorded in Asia are only 6 Myr old
4
. However, ana-
lyses of fossil clades have not resolved the issue of cetacean relations.
Proposed sister groups ranged from the entire artiodactyl order
5,6
,to
the extinct early ungulates mesonychians
7
, to an anthracotheroid
clade
8
(which included hippopotamids), to weakly supporting hip-
popotamids (to the exclusion of anthracotheres
9,10
).
The middle Eocene artiodactyl family Raoellidae
11–14
is broadly
coeval wit h the earliest cetaceans, and both are endemic to south
Asia. Raoellids, as a composite consisting of several genera, have been
added to some phylogenetic analyses
5,10
, but no close relation to
whales was found because raoellid fossils were essentially limited to
dental material
11–14
. We studied new dental, cranial and postcranial
material for Indohyus, a middle Eocene raoellid artiodactyl from
Kashmir, India (Fig. 1). All fossils of Indohyus were collected at a
middle Eocene bone bed extending for about 50 m at the locality
Sindkhatudi in the Kalakot region of Kashmir on the Indian side of
the Line of Control. Our analysis identifies raoellids as the sister
group to cetaceans and bridges the morphological divide that sepa-
rated early cetaceans from artiodacyls. This has profound implica-
tions for the character transformations near the origin of cetaceans
and the cladistic definition of Cetacea, and identifies the habitat in
which whales originated. Taken together, our findings lead us to
propose a new hypothesis for the origin of whales.
Cetaceans and raoellids are sister groups
To investigate the importance of raoellids in cetacean phylogeny, we
excluded raoellids from our initial phylogenetic analysis of artiodac-
tyls plus cetaceans. Our data set differed from previous analyses
10
by
the addition of several archaic anthracotheres, and some corrected
scores for pakicetid cetaceans. This analysis found stronger support
for hippopotamid–cetacean sister-group relations than the previous
analysis
10
, consiste nt with molecular studies
1–3
. However, the base of
the artiodactyl cladogram is poorly resolved (see Supplementary
Information for details on phylogenetic runs). In a second cladistic
analysis (Fig. 2), we added the raoellids Khirtharia and Indohyus as
well as several archaic ungulate groups (condylarths) and found that
raoellids and cetaceans are sister groups and that they are the basal
node in the Cetacea/Artiodactyla clade, consistent with some pre-
vious analyses that used different character sets
5,6
. Our analysis is the
first to show that raoellids are the sister group to cetaceans, resolving
the biogeographic conundrum and closing the temporal gap between
cetaceans and their sister. Relations between most artiodactyl families
higher in the tree are poorly resolved, and our data lack implications
for the relations between these families. Our analysis strongly argues
that raoellids and cetaceans are more closely related to each other
than either is to hippopotamids.
Indohyus shares with cetaceans several synapomorphies that are
not present in other artiodactyls. Most significantly, Indohyus has a
thickened medial lip of its auditory bulla, the involucrum (Figs 1 and
3), a feature previously thought to be present exclusively in cetaceans.
Involucrum size varies among cetaceans, but the relative thickness of
medial and lateral walls of the tympanic of Indohyus is clearly within
the range of that of cetaceans and is well outside the range of other
cetartiodactyls (Fig. 3). Other significant derived similarities between
Indohyus and cetaceans include the anteroposterior arrangement of
incisors in the jaw, and the high crowns in the posterior premolars.
Characterizing Cetacea
Until now, the involucrum was the only character occurring in
all fossil and recent cetaceans but in no other mammals
5,15,16
.
Identification of the involucrum in Indohyus calls into question
what it is to be a cetacean: it requires either that the concept of
Cetacea be expanded to include Indohyus or that the involucrum
cease to characterize cetaceans. We argue that the content
of Cetacea should remain stable and include Pakicetidae,
Ambulocetidae, Remingtonocetidae, Protocetidae, Basilosauridae,
Mysticeti and Odontoceti
6,7,9,10,17
. Thus, Cetacea remains a mono-
phyletic group, whereas Artiodactyla remains a paraphyletic group
(because Raoellidae are included but Cetacea are excluded).
An alternative classification would render both Cetacea and
Artiodactyla monophyletic by including Raoellidae in Cetacea and
1
Department of Anatomy, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272, USA.
2
School of Biomed ical Sciences, Kent State University, Kent, Ohio 44242,
USA.
3
Department of Geology and Geophysics, University of Wyoming, Laramie, Wyoming 82071, USA.
4
Department of Earth Sciences, Indian Institute of Technology, Roorkee,
Uttarakhand 247 667, India.
5
Wadia Institute of Himalayan Geology, Dehra Dun, Uttarakhand 248 001, India.
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by limiting Artiodactyla to those clades one node above the raoellid/
cetacean node (Fig. 2). We do not prefer this classification because it
causes instability by significantly altering the traditional content of
both Artiodactyla and Cetacea.
Characters identified as synapomorphies for Cetacea in some of
our most parsimonious trees include: long external auditory meatus,
double-rooted P3/, lack of P4/ protocone, M1-2/ metacones present
but small, and lack of M1-2/ hypocone. None of these features char-
acterize all modern and extinct cetaceans; the dental characters, for
instance, cannot be scored in toothless mysticetes. In addition, all of
these characters are found in some mammals unrelated to cetaceans.
We attach particular importance to two character complexes that
characterize basal cetaceans, constitute synapomorphic suites for the
order, and are of great functional importance. All fossil and recent
cetaceans differ from most other mammals in the reduction of crush-
ing basins on their teeth: there are no trigonid and talonid basins in
the lower molars, and the trigon basin of the upper molars is very
small (for example in pakicetids and ambulocetids) or absent.
Crushing basins are large in raoellids (Fig. 1a, b) and other basal
ungulates. This implies that a major change in dental function
occurred at the origin of cetaceans, probably related to dietary change
at the origin
18
. Reduced crushing basins also occur in mesonychids,
archaic ungulates long thought to be closely related to cetaceans.
However, mesonychian molars have wear facets very unlike those
of cetaceans
7,18
, whereas wear facets in raoellids are more similar to
wear facets in early cetaceans
14
.
The second character complex that identifies cetaceans is the shape of
the postorbital and temporal region of the skull. In early cetaceans, this
region is long and narrow
19
. This affects the sense organs: the olfactory
peduncle is long and narrow and the orbits are set close together near
the roof of the skull. It also affects oral function, the nasopharyngeal
duct is narrow, and the out-lever of the masticatory muscles is long,
increasing the closing speed of the jaws. We speculate that the import-
ance of different sense organs was related to these changes, or that
changing diet led to a change in food-processing organs.
dc
b
a
e
f
g
h
i
j
k
l
Involucrum
1 cm
1 cm
Cortical
Medullary
Crushing
basins
cavity
bone
Lateral
tympanic
wall
Middle ear
cavity
Tympanic bulla
Figure 1
|
Osteology of Indohyus and cross-sections of long bones of
Eocene cetartiodactyls. a
, b, Oblique lateral view of skull RR 208 (a) and
ventral view of skull RR 207 (
b). c–h, Posterior views of humerus (RR 149,
c) and femur (RR 101, d), plantar views of metacarpal (RR 138, e) and
proximal manual phalanx (RR 19,
f), dorsal view of astragalus (RR 224, g),
and posterior view of metatarsal (RR 139,
h). i–l, Histological mid-shaft
sections for humerus of the pakicetid Ichthyolestes (H-GSP 96227,
i),
humerus of Indohyus (RR 157,
j), femur of Indohyus (RR 42, k) and femur of
the artiodactyl Cainotherium (IVAU unnum,
l). Both scale bars are 1 cm; the
scale bar near
d goes with a–h, and that near l goes with i–l.
Arctocyon
Hyopsodus
Phenacodus
Meniscotherium
Eoconodon
Andrewsarchus
Hapalodectes
Dissacus
Ankalogon
Sinonyx
Pachyaena gigantea
Pachyaena ossifraga
Mesonyx
Synoplotherium
Harpagolestes
Khirtharia
Indohyus
Pakicetus
Ambulocetus
Rodhocetus
Artiocetus
Diacodexis
Gujaratia
Cebochoerus
Mixtotherium
Siamotherium
Amphirharagatherium
Gobiohyus
Homacodon
Bunomeryx
Microbunodon
Anthracokeryx
Elomeryx
Entelodon
Perchoerus
Tayassu
Sus
Choeropsis
Hippopotamus
Merycoidodon
Agriochoerus
Protoceras
Heteromeryx
Leptoreodon
Hypertragulus
Leptomeryx
Amphimeryx
Xiphodon
Eotylops
Poebrotherium
Cainotherium
Tragulus
Oth
e
r Artiodactyl
a
Mes
o
ny
chi
dae
Cetacea Raoellidae
Figure 2
|
Phylogeny of artiodactyls, cetaceans and archaic ungulates. The
figure shows a consensus cladogram produced by heuristic searches with
PAUP (random addition sequence, 1,000 repetitions), using a published
data set
10
. See Supplementary Information for further details.
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Indohyus was aquatic
Behaviourally, the earliest whales (pakicetids) were aquatic
waders
5,20–23
. This led us to investigate whether Indohyus was aquatic
too. Cortical bone thickness in secondarily aquatic tetrapods is com-
monly increased at the expense of the medullary cavity, a pattern
called osteosclerosis
24
. Osteosclerosis occurs in early whales
20,21,25
,
manatees
26,27
, sea otters
28
, Hippopotamus
29
, beavers
29
, pinnipeds
29
and Mesozoic marine reptiles
26,30
. Osteosclerosis provides ballast
that allows some aquatic taxa to be bottom walkers (hippopotamids)
and others to maintain neutral buoyancy in water (manatees)
30
.
Histological sections indicate that the limb bones of Indohyus are
also osteosclerotic (Fig. 1i–l), in a similar manner to those of paki-
cetid cetaceans. Our survey of cortical bone thickness in the limb
bones of terrestrial artiodactyls shows that this pattern is unusual
for that order: in mid-bone cross-sections of the femur, the medul-
lary cavity makes up between 0.60 and 0.75 of the width of the bone,
whereas in aquatic mammals the values are lower (Hippopotamus,
0.55; pakicetids and ambulocetids, 0.25–0.57). In Indohyus this ratio
is 0.42, suggesting that Indohyus was osteosclerotic and thus aquatic.
We interpret the limb osteosclerosis of Indohyus to be related to
bottom walking and not to slow swimming, because the limbs are
gracile and not modified into paddles.
To investigate further the hypothesis that Indohyus was aquatic, we
studied the stable isotopes of its enamel, a tissue relatively resistant
to preburial and postburial alteration of isotopic composition
31
.
Enamel d
18
O values are influenced by the oxygen isotope composi-
tion of the food and water ingested by an animal as well as by certain
physiological processes (such as sweating, panting and respiration)
32
.
For aquatic species, the flux of environmental water by means of
direct ingestion and transcutaneous exchange overwhelms all other
oxygen sources
33
and can cause the enamel d
18
O values of freshwater
taxa (for example Hippopotamus) to be 2–3% lower than those for
terrestrial mammals
33,34
. Mean d
18
O values for four individuals of
Indohyus are at least 2% lower than those for our comparative sample
of Eocene terrestrial and semi-aquatic mammals from formations of
India and Pakistan of similar or slightly older age (Fig. 4)
22,23
.
Although not representative of the specific deposits from which
Indohyus was collected, oxygen isotope values for each ecological type
from these sites (namely terrestrial, 24–28%; semi-aquatic, 23% or
less) are surprisingly consistent regardless of age or location (Fig. 4).
This suggests that temporal and spatial variation in environmental
isotope values was relatively minor and was most probably insuf-
ficient to account for the extremely low d
18
O we have reported for
Indohyus. We did not recover tooth material of other mammals at the
Indohyus site; until such material can be analysed, the most consistent
interpretation is that these low values are a result of the aquatic habits
of this species.
Supporting evidence for a semi-aquatic life of Indohyus comes
from examination of its inter-individual variation in d
18
O values.
The overwhelming influx and mitigating influence of isotopically
homogeneous environmental water causes the variation in individual
d
18
O values for semi-aquatic and aquatic species (s.d. , 0.5%)tobe
much lower than that of terrestrial species (s.d. . 1.0%)
33
. This line
of evidence is especially relevant in our study because it does not
require an approximation of the mean environmental isotope values
for a site through analysis of the associated fauna. Variation in d
18
O
values for Indohyus (s.d. 5 0.4%) is extremely low and when com-
pared with species of sufficient sample size (n . 3) it is similar to that
of the semi-aquatic archaeocete Pakicetus. Given that the influence of
physiological and environmental factors on body water d
18
O values is
more strongly felt at smaller body sizes
35
, this low level of variation is
particularly compelling for Indohyus, with a body mass of less than
50 kg.
To explore the diet of Indohyus we studied carbon isotopes.
Enamel d
13
C values are defined by the carbon isotope composition
of an animal’s diet and can be used to identify the food webs and
resources used by an animal
36
. The d
13
C values of primary producers
at the base of aquatic and terrestrial food webs overlap, but values for
freshwater phytoplankton are typically depleted in
13
C relative to
freshwater macrophytes
37
, and both types of aquatic producer are
depleted in
13
C relative to terrestrial C
3
plants
37–39
. Consumers for-
aging within food webs fuelled by freshwater phytoplankton (for
example freshwater and brackish-water foraging Eocene whales) typ-
ically have lower d
13
C values than species foraging on aquatic macro-
phytes
37
or on terrestrial resources
33
(Fig. 4). Enamel d
13
C values for
Indohyus are higher than those for most early cetaceans and are most
similar to the d
13
C values in enamel for terrestrial mammals from
early and middle Eocene deposits in India and Pakistan. Indohyus
could have been feeding on land or in water, but it was clearly eating
something different from archaeocetes such as Pakicetus and
Ambulocetus. If the large crushing basins in the molars of Indohyus
were used for processing vegetation, these d
13
C values in enamel
could come from the ingestion of terrestrial plants or aquatic macro-
phytes. Alternatively, a more ominivorous diet would suggest that
Indohyus might have foraged on benthic, aquatic invertebrates in
Indohyus
2.5
3.0
3.5
4.0
4.5
5.0 5.5
0
5
10
15
20
25
l
n [Width
oc
c
ip
ital co
n
dyl
e
s
(m
m
)
]
Tympanic thickness (medial/lateral walls)
Figure 3
|
Plot of the ratio of the thickness of the medial tympanic wall to
that of the lateral tympanic wall against the natural logarithm of the width
across occipital condyles, showing that the ratio in Indohyus is similar to
that in cetaceans.
In cetaceans (open squares), the medial tympanic wall is
inflated and called the involucrum, and the lateral tympanic wall is thinned
and called the tympanic plate. In artiodactyls (open triangles), the medial
and lateral tympanic walls are more similar in thickness, causing values on
the y axis to be closer to 1. See Supplementary Information for further
details.
20 22 24 26 28 30
–12
–8
–14
–10
–6
Indohyus
Freshwater and
brackish-water
Archaeocetes
Terrestrial
mammals
Brackish-water
Anthracobunids and
Khirtharia
δ
13
C
δ
18
O
Figure 4
|
Bivariate plot of d
18
O and d
13
C values for enamel samples of
early and middle Eocene mammals from India and Pakistan.
Results are
shown as means 6 s.d. for the sample population. Filled triangle, Indohyus;
open triangle, Khirtharia; open squares, terrestrial mammals; filled squares,
brackish-water anthracobunids; filled circles, freshwater/brackish-water
archaeocetes. See Supplementary Information for details.
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freshwater systems. Although we cannot exclude the possibility of
aquatic foraging by Indohyus, d
13
C values in enamel do suggest that
the diet of Indohyus differed significantly from that of Eocene whales.
A more refined interpretation of the dietary preferences of Indohyus
will require a study of tooth wear and tooth morphology.
Evolutionary hypothesis for whale origins
Indohyus was a small, stocky artiodactyl, roughly the size of the rac-
coon Procyon lotor (Fig. 5). It was not an adept swimmer; instead it
waded in shallow water, with its heavy bones providing ballast to keep
its feet anchored. Indohyus may have fed on land, although a special-
ized aquatic diet is also possible.
The modern artiodactyl morphologically most similar to Indohyus
is probably the African mousedeer Hyemoschus aquaticus.
Hyemoschus lives near streams and feeds on land, but flees into the
water when danger occurs
40
. Indohyus had more pronounced aquatic
specializations than Hyemoschus does, and it probably spent a con-
siderably greater amount of time in the water either for protection or
when feeding. As indicated by the evidence from stable isotopes,
Indohyus spent most of its time in the water and either came onshore
to feed on vegetation (as the modern Hippopotamus does) or foraged
on invertebrates or aquatic vegetation in the same way that the mod-
ern muskrat Ondatra does.
Raoellids are the sister group to cetaceans, and this implies that
aquatic habitats originated before the Order Cetacea. The great
evolutionary change that occurred at the origin of cetaceans is thus
not the adoption of an aquatic lifestyle. Here we propose that dietary
change was the event that defined cetacean origins; this is consistent
with the cranial and dental synapomorphies identified. Molars of
Indohyus are markedly different from those of pakicetids, and it is
widely assumed that pakicetids ate aquatic prey
18,23
.
Our working hypothesis for the origin of whales is that raoellid
ancestors, although herbivores or omnivores on land, took to fresh
water in times of danger. Aquatic habits were increased in Indohyus
(as suggested by osteosclerosis and oxygen isotopes), although it did
not necessarily have an aquatic diet (as suggested by carbon isotopes).
Cetaceans originated from an Indohyus-like ancestor and switched to
a diet of aquatic prey. Significant changes in the morphology of the
teeth, the oral skeleton and the sense organs made cetaceans different
from their ancestors and unique among mammals.
METHODS SUMMARY
We chose an existing character matrix
10
as the basis for our phylogenetic analysis.
We corrected scores of some of the taxa, and made some changes in the taxa
included. Details on these taxa, the rationale for using them, and their scores are
given in Methods and in Supplementary Table 1.
Tympanic wall thickness was investigated to address the presence of the
involucrum quantitatively. We calculated the ratio of medial tympanic wall
thickness divided by lateral tympanic wall thickness (see Methods and
Supplementary Table 2). Bone histology was studied to investigate the presence
of osteosclerosis. It was quantified as the ratio of medullary cavity width
divided by bone width in the mediolateral plane (see Methods and
Supplementary Table 3).
For analysis of stable isotopes, we prepared powders by following published
methods (see Methods and Supplementary Tables 4 and 5). Multiple samples
were collected for each species to provide an estimate of population means for
carbon and oxygen isotope values
33
. About 5 mg of enamel powder was collected
from each specimen (tooth) for study of carbon and oxygen isotope values.
Most fossils of Indohyus were collected by the late Indian geologist A. Ranga
Rao, who discovered the locality about 25 years ago (acronym RR); additional
fossils were collected by S.B. and B.N.T. at the same locality (acronym IITR-SB-
Kal-S).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 26 June; accepted 3 October 2007.
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10 cm
Figure 5
|
Skeletal reconstruction of Indohyus. Hatched elements are
reconstructed on the basis of related taxa.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank the late F. Obergfell for presenting us with the
sediment blocks containing Indohyus fossils collected by A. Ranga Rao for
preparation and study; D. S. N. Raju and N. Raju for facilitating our research;
B. Armfield, R. Conley and A. Maas for fossil preparation; J. Dillard for preparing
Fig. 5; and J. Geisler and J. Theodor for providing additional information about their
cladistic analyses. Laboratory research was funded by the National Science
Foundation (NSF)
–
Earth Sciences (grants to J.G.M.T. and M.T.C.). Collaborative
work was funded by the Indian Department of Science and Technology (to S.B.)
and the NSF
–
International Division (to J.G.M.T.) under the Indo-US Scientific
Cooperation Program. Laboratory analyses were supported by the Skeletal Biology
Research Focus Area of Northeastern Ohio Universities College of Medicine.
Author Contributions J.G.M.T. was responsible for anatomical and systematic
study, and scientific synthesis, L.N.C. for systematic and bone density study,
M.T.C. for the study of stable isotopes, and S.B. and B.N.T. for geological study and
collecting of Indohyus and comparative fossil samples.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to J.G.M.T. (thewisse@neoucom.edu).
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METHODS
Systematic study. We chose an existing, published character matrix
10
as the basis
for our phylogenetic analysis because this matrix is rich in characters and con-
tains most relevant taxa. From this matrix we deleted those artiodactyls and
cetaceans that are geologically young or modern and are well represented by
fossil relatives (Odocoileus, Bos, Ovis, Remingtonocetus, Protocetus, Georgiacetus,
Basilosaurus, Balaenoptera, Physeter, Tursiops, Delphinapterus, Camelus and
Lama), as well as perissodactyls and non-ungulate taxa.
To this matrix, we added the anthracotheres Siamotherium, Anthracokeryx and
Microbunodon, because they are near the base of the anthracotheroid clade
(Anthracotheriidae plus Hippopotamidae) and are sometimes thought to be
close to early whales
4
. Scores for these taxa, and the sources on which we based
the scores, are listed in Supplementary Table 1 (refs 4, 6, 41–46).
We chose Gujaratia pakistanensis (formerly Diacodexis pakistanensis)
47
as out-
group for the analyses of cetaceans plus artiodactyls, and we chose Arctocyon and
Hyopsodus as outgroups for the (second) analysis that included all taxa (Fig. 2).
We corrected some of the scores for Pakicetidae
10
, because new fossils have
been published for this family, in particular cranial material
19
and postcranial
material
21
. Corrected scores for pakicetids are also listed in Supplementary Table
1 and were based on original material in the Howard-Geological Survey of
Pakistan (H-GSP) collections, currently curated by J.G.M.T.
Raoellidae have been included in several previous phylogenetic analyses relat-
ing to early whales
5,6
. These authors based raoellid scores on Khirtharia and
Indohyus. In the present analyses we have split scores for these animals, with
Khirtharia scores based mostly on published H-GSP material and one unpub-
lished skull (H-GSP 1979; dentition published
48
, specimen now lost). Scores for
Indohyus are based on the material in the RR and IITR-SB collections; all raoellid
scores are listed, with the specimen number of the fossil on which the score was
based, in Supplementary Table 1.
Study of tympanic walls. Tympanic wall thickness was investigated to address
the presence of the involucrum quantitatively. The involucrum is the thickened
medial wall of the tympanic bone (the ossified wall of the middle ear cavity). The
lateral tympanic wall of cetaceans is reduced in thickness (the tympanic plate).
To quantify these differences in tympanic walls, we calculated the ratio of medial
tympanic wall thickness to the lateral tympanic wall thickness. Lateral tympanic
wall thickness was measured with a micrometer (Dyer gauge) just inferior to
the tympanic ring, and medial tympanic wall thickness was measured directly
across from this site on the other (medial) side of the middle ear cavity (see
Supplementary Table 2).
Bone histology. Bone histology was studied to investigate the presence of osteo-
sclerosis. Osteosclerosis is the thickening of the cortical bone. It was quantified as
the ratio of medullary cavity width divided by bone width in the mediolateral
plane, because left and right cortical thickness plus medullary cavity thickness
equals bone width (see Supplementary Table 3). Measurements were taken on
the femur with callipers.
Fossil limb shaft fragments were embedded in Buehler low-viscosity epoxy
resin and sectioned with a diamond saw. Sections were mounted on frosted glass
slides by using epoxy resin. Mounted sections were then ground down and
polished to a thickness of about 75 mm by using a precision grinder with 600,
800 and 1,200 grit paper
20
(Fig. 1i–l).
Study of stable isotopes. For the analysis of stable isotopes, three or more speci-
mens of each species were analysed (when available; see Supplementary Tables 4
and 5) to provide a robust estimate of the population mean and s.d. for carbon
and oxygen isotope values
33
. About 5 mg of enamel powder was collected from
each specimen, either by drilling directly from the tooth or by grinding enamel
chips in an agate mortar and pestle. Before collection, contaminants were
removed by abrading the outer surface of the specimen.
Preparation of powders for analysis of stable isotopes followed published
methods
31
. Powders were first transferred to 1-ml microcentrifuge vials and then
soaked sequentially overnight in about 0.20 ml of a sodium hypochlorite solu-
tion (1–2 g dl
21
) and then in about 0.20 ml of calcium acetate buffered acetic acid
(pH about 5.1). On addition of each reagent, samples were agitated for 1 min on
a Vortex Genie vortex mixer. After each soak, the supernatant was removed by
aspiration and the residual powder was rinsed five times with deionized water.
Samples were then freeze-dried overnight and about 1.5 mg of powder from each
was weighed into individual test tubes for analysis on a Thermo-Finnigan gas
bench autosampler attached to a Thermo Finnigan Delta
Plus
XP continuous-flow
isotope-ratio mass spectrometer at the University of Wyoming Stable Isotope
Facility.
All values for stable isotopes are reported in delta (d) notation, using the
equation d(%) 5 1,000 3 (R
sample
/R
standard
2 1), where R
sample
is the observed
isotope ratio of the sample (
13
C/
12
Cor
18
O/
16
O) and R
standard
is the accepted
ratio for an appropriate international standard (Vienna Pee Dee belemnite for
d
13
C; Vienna Standard Mean Ocean Water for d
18
O). Analytical precision is
typically better than 0.1% for d
13
C values and 0.2% for d
18
O values (61s).
41. Ducrocq, S. The late Eocene Anthracotheriidae (Mammalia, Artiodactyla) from
Thailand. Palaeontographica A 252, 93
–
140 (1999).
42. Ducrocq, S. Unusual dental morphologies in late Eocene anthracotheres
(Artiodactyla, Mammalia) from Thailand: dental anomalies and extreme
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¨
ont. MH 4, 199
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212 (1999).
43. Suteethorn, V., Buffetaut, E., Helmcke-Ingavat, R., Jaeger, J. J. &
Jongkanjanasoontorn, Y. Oldest known Tertiary mammals from South East Asia:
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couverte d’un cra
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97
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115 (2004).
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doi:10.1038/nature06343
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